EP0804474B1 - Identifizierung, kwantifizierung und reinigung von insektiziden proteinen aus bacillus thuringiensis - Google Patents

Identifizierung, kwantifizierung und reinigung von insektiziden proteinen aus bacillus thuringiensis Download PDF

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EP0804474B1
EP0804474B1 EP94919536A EP94919536A EP0804474B1 EP 0804474 B1 EP0804474 B1 EP 0804474B1 EP 94919536 A EP94919536 A EP 94919536A EP 94919536 A EP94919536 A EP 94919536A EP 0804474 B1 EP0804474 B1 EP 0804474B1
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process according
eluent
toxins
bacillus thuringiensis
strain
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EP0804474A1 (de
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Marianne Pusztai-Carey
Paul Richard Carey
Makoto Yaguchi
Timothy Lessard
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National Research Council of Canada
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/50—Isolated enzymes; Isolated proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
    • C07K14/325—Bacillus thuringiensis crystal peptides, i.e. delta-endotoxins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient

Definitions

  • the invention relates to the identification, quantitation and purification of insecticidal proteins from Bacillus thuringiensis, in particular from multigene strains of Bacillus thuringiensis.
  • Bt Bacillus thuringiensis
  • Bt strains with different insect host toxicity spectra have been identified. Numerous strains are active against larvae of certain members of the Lepidoptera (Bt strains active against over 100 species of lepidopterans have been identified to date), but strains showing toxicity against dipteran or coleopteran species are also known.
  • Bt parasporal inclusions have proven to be a valuable alternative to conventional insecticides. They are highly toxic to the target insects and harmless to the environment owing to their specificity.
  • Bt produces several types of toxins, the precise biochemical nature of which may vary from strain to strain. Some of them, in particular ⁇ - and ⁇ -exotoxins, are toxic to a variety of insect orders or to many cell types.
  • the parasporal crystal inclusion toxins also called ⁇ -endotoxins
  • Bt crystalline inclusions dissolve in the larval midgut and rapidly undergo proteolytical conversion into smaller toxic polypeptides (in the 23-80 kD molecular weight range) in the insect midgut.
  • the produced toxin species interact with midgut epithelium cells of the host insect, generating pores in the cell membrane and disturbing the osmotic balance.
  • Bt is provided with a surprisingly large and variable family of insecticidal proteins.
  • Data obtained using several experimental methods indicate that crystal protein genes in many subspecies of Bt that are toxic to lepidopterans are located on one or more large plasmids; in some subspecies, the gene may be located on the chromosome.
  • the fact that the genes for the Bt protoxins are usually plasmid borne has made Bt a favourite candidate for genetic manipulations. This has resulted recently, among other things, in generating insect-resistant transgenic crop plants capable of expressing Bt crystal protein genes.
  • Bt strains contain several closely related genes coding for protoxins.
  • Bt var. kurstaki NRD-12 strain is a three-gene strain. The presence of several such genes results in the production by a single Bt strain of several protoxins closely related by their amino acid sequences. Enzymatic action of proteolytic enzymes on such a mixture of protoxins, either in vitro or in the insect midgut, produces several toxins which may only differ by a few amino acid residues, which makes the obtained mixture of toxins difficult to separate. However, since these small differences frequently occur in key regions of the toxin sequence, they may result in significantly different toxicities towards a selected insect target.
  • composition of the mixture of toxins produced by a multigene strain of Bt or the expression level of the individual toxins may vary with fermentation conditions, so may its relative toxicity to various insects, and, as a consequence, the host range of the multigene Bt strain. It becomes therefore important to monitor and quantitate different toxins present in endotoxin crystals, in order to optimize the production of the most wanted toxin from a multigene producer.
  • Bt strains have been proposed. They involve, for example, the use of flagellar antibodies, probing for the genes with DNA probes or measuring the level of RNA production. These methods, although useful for characterization and classification purposes, are not satisfactory for quantitation of gene expression and monitoring the viability of a given strain or producing individual toxin standards for analyzing insecticidal activity, synergism, membrane studies or insect resistance.
  • the toxin mixture is denatured in urea and re-digested by trypsin for peptide mapping using reverse-phase HPLC (the peptides were solubilized in acid and eluted in acid/acetonitrile mixtures). As mentioned, above, this destroys the biological activity of the toxin.
  • the resultant peptide mapping using single gene standards to interpret the results from multi-gene strains, failed to recognize the presence of the cryIA(c)[6.6] gene product in the strain kurstaki HD-1. This gene and its protein toxin is an important component, shown to be present by other researchers.
  • the present invention provides a process for identifying a protoxin expressed by a Bacillus thuringiensis gene, which process comprises
  • aughter toxin may cover a mixture of protoxins or an individual protoxin, depending on the starting material.
  • a mixture of protoxins is involved
  • a single gene strain of Bt is used, a single protoxin is involved.
  • the invention can be used to characterize a single toxin obtained from a single-gene strain of Bt.
  • the strain of Bt is also characterized.
  • the level of expression of known toxins from a known Bt strain can be determined and the viability of the strain can be monitored.
  • One embodiment according to the invention comprises typically the following steps:
  • the material digested by proteolytic enzymes is normally crystalline endotoxin purified from crude fermentation mixtures, such a purification, although preferred, is not essential. Recognizable peaks characteristic of toxins in the anion exchange HPLC elution pattern can be also obtained using a crude Bt material separated from the fermentation broth prior to enzymatic digestion.
  • the purified crystals or washed crude material are then subjected directly to hydrolysis with a proteolytic enzyme, such as trypsin, chymotrypsin or elastase. Trypsin is the preferred enzyme. Also insect gut juices, such as gut juice from silk worm (Bombyx mori) are effective. Each particular enzyme gives a slightly different set of toxins, since each has a unique specificity with respect to endotoxin proteins. Commercially available preparations of enzymes may be used. The concentration range of the enzyme is from 0.1 to 2 mg/mL, preferably 1 mg/mL.
  • the temperature of digestion of the endotoxin with the proteolytic enzyme is not critical and may vary from 20 to 40°C, preferably, about 37°C.
  • the duration of hydrolysis is also not critical but should be long enough to assure a nearly complete liberation of toxins under given pH, temperature and enzyme concentration conditions. For trypsin digestion, the time of digestion is about 10 minutes to 12 hours.
  • the hydrolysis can be carried out in an unbuffered or buffered solution having pH in the specified range.
  • suitable solutions are 3-cyclohexylamino-1-propanesulfonic acid/NaOH buffer (CAPS) borate buffer or unbuffered solution of NaOH. Not all solutions are equally efficient. Since the toxins released during hydrolysis are apparently hydrophobic and poorly soluble in water, it is preferable that the solution contains components which assist solubilization of the toxin protein molecules.
  • One example of such a component is 3-cyclohexylamino-1-propanesulfonic acid (CAPS).
  • the preferred solution is 0.1 M CAPS/NaOH buffer having pH 10.5.
  • the solid components of the reaction mixture are removed, for example by centrifugation and filtration. Samples of the filtrate (supernatant) are used for HPLC analysis. The pellet should not contain any recognisable inclusion body by phase contrast microscope (as a sign of complete digestion).
  • the HPLC separation of the products of proteolytic hydrolysis provides a fingerprint by which the toxins can be identified and quantified.
  • the separation must be conducted at pH not lower than about 10, preferably 10.5 to 11.5.
  • pH should not be higher than about 12, to avoid the denaturation of the toxins and to assure the proper operation of the column.
  • the molecules of toxins are negatively charged and the separation is carried out as a liquid-solid anion exchange chromatography.
  • the use of HPLC technique allows the achieving of the separation in short periods of time.
  • the separation of the toxins according to the invention can be carried out at any value of pH in the indicated broad range, increasing the pH above the preferred range of 10.5 to 11.5 unnecessarily increases the danger of denaturation of the separated proteins and irreversible damages to the column.
  • decreasing the pH below 10.5 increases the danger of toxins being precipitated in the HPLC column; at least partially, which adversly affects the reliability of quantitative determinations.
  • the pH range of about 10.5 to 11.5 is considered to be the optimum.
  • the elution of the toxins from the column is carried out at room temperature employing a suitable buffer, in an increasing gradient of a salt, such as sodium chloride.
  • a suitable buffer in an increasing gradient of a salt, such as sodium chloride.
  • the employed buffer is in first place responsible for maintaining the required pH of the eluent.
  • the conditions of salt gradient are specific for the column used. These conditions are normally achieved by employing a series of buffers having increasing concentration of the salt and introduced in a predetermined order and at a predetermined time.
  • the salt used in the separation should bind rather strongly to the column, but should not bind to or otherwise adversly affect the separated proteins.
  • suitable salts include sodium chloride, potassium chloride, ammonium chloride and sodium acetate. Bromides and iodides may also be used. Sodium chloride is preferred. Some salts, for instance calcium salts, bind to some proteins and affect the properties of the protein. These salts are therefore not suitable. Selection of a suitable salt will present no problem to a person skilled in the art.
  • the elution from the chromatographic column is effected, immediately after hydrolysis, in two stages.
  • a first eluent is 0.05 M CAPS/NaOH buffer, pH 11.5, followed by a second eluent of 0.05 M CAPS/NaOH buffer and 0.5 M NaOl, pH 11.5.
  • the two eluents are simultaneously introduced into the column in such a manner that the amount of the second eluent increases linearly from 0 to 33 % over a period of 25 to 40 minutes, depending upon the nature of the sample. This step was followed by a 20-50 minute isocratic period while the toxinx are eluting.
  • Parts of the eluate corresponding to candidate peaks may be collected, concentrated and reinjected to establish identity and purity of the separated toxins.
  • an active, native toxin may be isolated and collected.
  • the method of rapid identification, quantitation and purification of toxins resulting from proteolytic hydrolysis of protoxins of endotoxin crystals is of considerable interest for the art. It provides means of screening and testing new Bt isolates by a rapid comparison against known strains. It also allows experiments in the manipulation of fermentation conditions to optimise the production of the most wanted toxin from multigene strains. It also enables producers to characterize the Bt toxins produced by competitors' strains. Since separated toxins may be collected in pure and active form, the method will permit testing of individual purified toxins and mixtures of individual toxins prepared in a specified and controlled manner. Although of particular interest for multigene strains of Bt, the method has obviously similar application for single gene strains of Bt and for analysis of inclusion bodies from cloned Bt genes, e.g. in E. coli.
  • the separation was carried out using Waters 990 solvent delivery system equipped with an automatic injector and a photodiode array detector.
  • Protein PAK DEAE 5PW anion exchange column (7.5 x 75 mm analytical or 21.5 x 150 mm semipreparative, Waters) was used; injection volume - 1-20,000 ⁇ L; flow rate - 4 mL/min.
  • Figure 2 shows a chromatogram of the tryptic digest under these gradient conditions, where the peaks of separated toxins are marked as B, C and D. Peak B corresponds to the cryIA(a)[4.5] gene toxin, peak C corresponds to the cryIA(b)[5.3] gene product and peak D represents the cryIA(c)[6.6] gene product.
  • the condition of the column or very slight changes of pH or salt concentration can result in the shifting of the retention time of certain proteins by several minutes. As can be seen from Figure 2 the separation takes place during approximately the first 60 minutes of elution. The following elution is for cleaning and regeneration of the column for subsequent separations.
  • Parts corresponding to B, C and D candidate peaks were collected from the HPLC eluate, concentrated using 8 kDa polyethylene glycol/3.5 kDa cut-off dialysis tubing and reinjected into the HPLC column to establish purity.
  • the respective chromatograms are shown in Figures 3, 4 and 5. With repeated injection-isolation procedure 100% purity of the isolated toxins can be achieved.
  • Toxins isolated and purified as described above were cleaved using CNBr and the resulting fragments were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE).
  • SDS-PAGE SDS-polyacrylamide gel electrophoresis
  • the gel was electroblotted onto polyvinylidene difluoride membrane.
  • the peptide bands in the membrane were stained, the visualized peptide bands were excised and candidate fragments were sequenced by an Applied Biosystems 475A protein sequencing system comprising a 470A gas phase sequencer equipped with an on-line 120A PTH analyser with a 900A control/data analysis module.
  • Figure 10 shows the sequences of the three toxins activated by trypsin and aligned according to identical sequence segments, identified respectively as cryIA(a) [4.5], cryIA(b) [5.3] and cryIA(c) [6.6].
  • the sequences underlined in Figure 10 correspond to peptides which have been isolated and sequenced and most of which contain amino acid sequences unique to only one protein.
  • FIG. 6 shows a chromatogram of the tryptic digest under these gradient conditions, where peaks 10, 11 and 12, appearing approximately between 22 and 28 minute of the elution, correspond to separated toxins being cryIA(a) [4.5], cryIA(b) [5.3] and cryIA(c) [6.6] gene products.
  • the separation takes place under a linear gradient of sodium chloride increasing from 0 to 0.5M during 35 minutes.

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Claims (27)

  1. Verfahren zur Identifizierung eines Protoxins, das von einem Bacillus thuringiensis-Gen exprimiert wird, welches Verfahren umfaßt
    Hydrolyse eines Bacillus thuringiensis-Protoxin enthaltenden Materials mit einem proteolytischen Enzym in einer wäßrigen Suspension bei einem pH im Bereich von 10 bis 12, um ein solubilisiertes Tochtertoxin zu bilden,
    Unterwerfen des Tochtertoxins einer Hochleistungs-Anionenaustauschflüssigkeitschromatographie bei einem im wesentlichen konstanten pH im Bereich von 10 bis 12 und unter wäßrigen Bedingungen, welche entsprechen der Verwendung eines ersten Elutionsmittels, das nur einen Puffer enthält, und der allmählichen Einführung im Laufe eines vorherbestimmten Zeitraums von mindestens einem weiteren Elutionsmittel, das den Puffer und ein geeignetes Salz enthält, wobei die Änderungen der Konzentration des Salzes im Elutionsmittel mit der Zeit so sind, daß das Tochtertoxin in biologisch aktivem Zustand von anderen Hydrolyseprodukten abgetrennt wird, und
    Identifizierung und erforderlichenfalls Quantifizierung des Protoxins anhand der chromatographischen Signale, die von dem Tochtertoxin erzeugt werden.
  2. Verfahren nach Anspruch 1, worin das Gen von einem Stamm von Bacillus thuringiensis exprimiert wird und das Verfahren zur Charakterisierung des Stammes eingesetzt wird.
  3. Verfahren nach Anspruch 2, worin der Stamm von Bacillus thuringiensis ein Multigen-Stamm ist und das Verfahren eingesetzt wird zur Charakterisierung des Stammes durch Auftrennung individueller Tochtertoxine, welche von den Protoxinen, die von jedem jeweiligen Gen exprimiert werden, erhalten werden.
  4. Verfahren nach Anspruch 3, worin der Stamm ein bekannter Stamm ist und das Verfahren eingesetzt wird, um das Expressionsniveau des Gens und die Lebensfähigkeit des Stammes zu bestimmen.
  5. Verfahren nach Anspruch 1, worin das Protein-Protoxin von einem klonierten Bacillus thuringiensis-Gen exprimiert wird.
  6. Verfahren nach Anspruch 1, welches ferner den Schritt der Isolierung und Reinigung eines individuellen Toxins in biologisch aktivem Zustand umfaßt.
  7. Verfahren nach Anspruch 1, worin das Protoxin-enthaltende Material Parasporen-Kristalle von Bacillus thuringiensis darstellt.
  8. Verfahren nach Anspruch 1, worin das Protoxin-enthaltende Material eine rohe Fermentationsmischung von Bacillus thuringiensis, abgetrennt von der Fermentationsbouillon, darstellt.
  9. Verfahren nach Anspruch 1, worin das proteolytische Enzym aus der Gruppe aus Trypsin, Chymotrypsin, Elastase und Insektenverdauungssäften ausgewählt ist.
  10. Verfahren nach Anspruch 9, worin das proteolytische Enzym Trypsin ist.
  11. Verfahren nach Anspruch 10, worin die Hydrolyse direkt bei einem pH von etwa 10,5 durchgeführt wird.
  12. Verfahren nach Anspruch 11, worin die Hydrolyse in 0,1 M 3-Cyclohexylamino-1-propansulfonsäure/NaOH-Puffer durchgeführt wird.
  13. Verfahren nach Anspruch 11, worin die Hydrolyse bei einer Temperatur von etwa 20 bis etwa 40°C durchgeführt wird.
  14. Verfahren nach Anspruch 12, worin die Chromatographie bei einem pH von 10,5 bis 11,5 durchgeführt wird.
  15. Verfahren nach Anspruch 14, worin die Chromatographie in 0,05 M 3-Cyclohexylamino-1-propansulfonsäure/NaOH-Puffer durchgeführt wird.
  16. Verfahren nach Anspruch 13, worin das Salz Natriumchlorid ist.
  17. Verfahren nach Anspruch 1, worin das Anionenaustauscher ein schwacher Anionenaustauscher ist.
  18. Verfahren nach Anspruch 17, worin der Anionenaustauscher Diethylaminoethylpoly(methylmethacrylat) ist.
  19. Verfahren nach Anspruch 1, worin der Anionenaustauscher ein starker Anionenaustauscher ist.
  20. Verfahren nach Anspruch 19, worin der Anionenaustauscher Trimethylammoniummethylgruppen enthält.
  21. Verfahren nach Anspruch 1, worin die Chromatographie bei einem pH ausgeführt wird, der im wesentlichen identisch mit dem pH ist, bei dem die Hydrolyse durchgeführt wird.
  22. Verfahren nach Anspruch 21, worin die Lösung nach der Hydrolyse direkt einer Chromatographie unterworfen wird.
  23. Verfahren nach Anspruch 1, worin die Signale der Hochleistungsflüssigkeitschromatographie identifiziert werden durch Vergleich mit chromatographischen Signalen, welche erhalten werden durch Unterwerfen eines Protoxins, das von einem bekannten Bacillus thuringiensis-Gen exprimiert wird, einer Hydrolyse mit demselben proteolytischen Enzym unter denselben Bedingungen, gefolgt von Anionenaustausch-Hochleistungsflüssigkeitschromatographie unter denselben Bedingungen.
  24. Verfahren nach Anspruch 1, worin das Protoxin-enthaltende Material mit Trypsin in 0,1 M 3-Cyclohexylamino-1-propansulfonsäure/NaOH-Puffer mit einem pH von 10,5 hydrolysiert wird, die Lösung nach der Hydrolyse direkt einer Chromatographie auf Diethylaminoethylpoly(methylmethacrylat) unterworfen wird, das erste Elutionsmittel 0,05 M 3-Cyclohexylamino-1-propansulfonsäure/NaOH-Puffer mit einem pH von 10,5 ist, das zweite Elutionsmittel derselbe Puffer mit 0,17 M Natriumchlorid ist, die beiden Elutionsmittel gleichzeitig auf solche Weise eingeführt werden, daß die Menge des zweiten Elutionsmittels im Verlaufe eines Zeitraums von etwa 20 Minuten linear von 0 auf 100% zunimmt, das dritte Elutionsmittel derselbe Puffer mit 0,5 M Natriumchlorid ist, die Einführung des dritten Elutionsmittels etwa 15 Minuten nachdem die Menge des zweiten Elutionsmittels 100% erreicht hat beginnt und die Menge des dritten Elutionsmittels im Verlaufe eines Zeitraums von etwa 15 Minuten linear von 0 auf 10% zunimmt.
  25. Verfahren nach Anspruch 1, worin das Protoxin-enthaltende Material mit Trypsin in 0,1 M 3-Cyclohexylamino-1-propansulfonsäure/NaOH-Puffer mit einem pH von 10,5 hydrolysiert wird, die Lösung nach der Hydrolyse direkt einer Chromatographie auf einem Harz, das Trimethylammoniummethylgruppen enthält, unterworfen wird, das erste Elutionsmittel 0,05 M 3-Cyclohexylamino-1-propansulfonsäure/NaOH-Puffer mit einem pH von 10,5 ist, das zweite Elutionsmittel derselbe Puffer mit 0,5 M Natriumchlorid ist, die beiden Elutionsmittel gleichzeitig auf solche Weise eingeführt werden, daß die Menge des zweiten Elutionsmittels im Verlauf eines Zeitraums von etwa 35 Minuten linear von 0 auf 100% zunimmt.
  26. Verfahren nach Anspruch 25, worin das zweite Elutionsmittel im Verlauf eines Zeitraums von mehr als etwa 30 Minuten eingeführt wird.
  27. Verfahren nach Anspruch 1, worin das Protoxin-enthaltende Material mit Trypsin in 0,1 M 3-Cyclohexylamino-1-propansulfonsäure/NaOH-Puffer mit einem pH von 10,5 hydrolysiert wird, die Lösung nach der Hydrolyse direkt einer Chromatographie auf einem Harz, das Trimethylammoniummethylgruppen enthält, unterworfen wird, das erste Elutionsmittel 0,05 M 3-Cyclohexylamino-1-propansulfonsäure/NaOH-Puffer mit einem pH von 11,5 ist, das zweite Elutionsmittel derselbe Puffer mit 0,5 M Natriumchlorid ist, die beiden Elutionsmittel gleichzeitig auf solche Weise eingeführt werden, daß die Menge des zweiten Elutionsmittels im Verlauf eines Zeitraums von etwa 40 Minuten linear von 0 auf 33% zunimmt.
EP94919536A 1990-03-14 1994-06-22 Identifizierung, kwantifizierung und reinigung von insektiziden proteinen aus bacillus thuringiensis Expired - Lifetime EP0804474B1 (de)

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US49345390A 1990-03-14 1990-03-14
US83696792A 1992-02-19 1992-02-19
US08/102,491 US5356788A (en) 1990-03-14 1993-08-05 Isolation, quantitation and purification of insecticidal proteins from Bacillus thuringiensis
PCT/CA1994/000346 WO1995035317A1 (en) 1990-03-14 1994-06-22 Identification, quantitation and purification of insecticidal proteins from bacillus thuringiensis

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JPH0618973B2 (ja) * 1988-01-20 1994-03-16 日本石油化学株式会社 熱可塑性樹脂組成物およびその製造方法
DE3923831A1 (de) * 1989-07-19 1991-01-31 Hartmann & Braun Ag Interferometrische einrichtung
JPH0357941A (ja) * 1989-07-27 1991-03-13 Asahi:Kk 摩擦試験機
JPH03244657A (ja) * 1990-02-22 1991-10-31 Polyplastics Co ポリエステル樹脂組成物及び成形品
JP2928318B2 (ja) * 1990-03-12 1999-08-03 ポリプラスチックス株式会社 ポリエステル樹脂組成物及び成形品
US5356788A (en) * 1990-03-14 1994-10-18 Her Majesty The Queen In Right Of Canada, As Represented By The National Research Council Of Canada Isolation, quantitation and purification of insecticidal proteins from Bacillus thuringiensis

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JPH10501410A (ja) 1998-02-10
GR3030216T3 (en) 1999-08-31
DE69416879D1 (de) 1999-04-08
AU7066294A (en) 1996-01-15
EP0804474A1 (de) 1997-11-05
WO1995035317A1 (en) 1995-12-28
ES2127930T3 (es) 1999-05-01
AU688335B2 (en) 1998-03-12
DK0804474T3 (da) 1999-10-04
DE69416879T2 (de) 1999-07-08

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